A. Field of the Invention
The present invention describes a shortened process for production of beer, brewer's yeast strains having .alpha.-acetolactate decarboxylase activity (EC 4.1.1.5.) (later .alpha.-ALDC), the method for the construction of such yeast strains and the recombinant DNA cloning vectors used.
The conventional brewing process can be shortened by accelerating either the primary fermentation or the secondary fermentation, or by producing beer in a continuous process. Of the different stages in the production of beer, the secondary fermentation, which is required for flavour maturation, takes the longest time (2-3 weeks) and requires much refrigerated space. Accelerating the costly flavour maturation is therefore more important than the speeding up of other stages of the process. A brewer's yeast capable of accelerated flavour maturation would have considerable economic value in the production of beer.
During the primary fermentation, yeast forms .alpha.-acetolactate, which is an intermediate in the biosynthesis of valine. Part of the .alpha.-acetolactate, not used in the synthesis of valine, leaks out of the yeast cell to the fermenting beer. In the beer, diacetyl is formed from this .alpha.-acetolactate by a spontaneous reaction. During secondary fermentation, diacetyl is enzymatically reduced to acetoin by the yeast (FIG. 1).
The degree of maturity of beer is determined in practice by its content of diacetyl, which is a typical aroma compound of butter. The taste and smell of diacetyl can be detected at a very low level and most people find it very obnoxious in beer. In light lager beers, the taste threshold value of diacetyl is between 0.02-0.05 mg/l. The taste threshold value of acetoin in beer is markedly higher (50 mg/l) than that of diacetyl.
B. Description of the Prior Art
Certain taxa of bacteria, e.g. enterobacteria (Aerobacter, Klebsiella, Enterobacter) and the Lactobacillus, Bacillus and Streptococcus bacteria used traditionally in the food industry have been found to possess the .alpha.-ALDC enzyme (EC 4.1.1.5.), which catalyzes the decarboxylation of .alpha.-acetolactate into acetoin (Godtfredsen et al., 1983). Klebsiella has been shown to be one of the best .alpha.-ALDC producers of the bacteria studied. The enzyme has not been found in organisms taxonomically higher than bacteria.
In laboratory experiments .alpha.-ALDC enzyme preparations have been added to freshly fermented beer, which resulted in the removal of diacetyl and its precursor from the beer within 24 h (Godtfredsen and Ottesen, 1982). Consequently, the time required for the maturation of beer was shortened. The quality of beer matured using .alpha.-ALDC was not found to be different from that of beer matured in the traditional way. It is to be noted that the use of additives in the production of beer is prohibited by food regulations in some countries. In addition, the commercial enzyme preparations are unspecific and thus contain also other enzyme activities as impurities. The addition of enzyme during the brewing process also increases the risk of contamination.
It is possible that accelerated secondary fermentation could be achieved not only by adding .alpha.-ALDC enzyme to young beer, but also by isolating the gene coding for this enzyme (later .alpha.-ald) from a suitable donor organism and expressing this gene in yeast. A yeast expressing this gene will produce the enzyme .alpha.-ALDC during primary fermentation and excess .alpha.-acetolactate would be decarboxylated directly to acetoin. Thus the addition of enzyme preparations to beer would become unnecessary. Using a brewer's yeast which carries only the specific gene would thus result in a rapid and clean process with no additives required.
The gene coding for .alpha.-ALDC has been isolated from Enterobacter aerogenes strain IFO 13534 (Sone et al., 1987). The gene was shown to be present on a 1.7 kb DNA-fragment but the localization of the gene in this DNA fragment was not given nor was its nucleotide sequence determined. In addition, no description of the methods used to express this gene in yeast was provided.
Although some bacterial DNA sequences carrying a coding region for a protein e.g. .beta.-lactamase can be expressed in yeast, the expression is in most of the cases insufficient or no expression is observed. To be efficiently expressed in yeast, the foreign gene has to be coupled to yeast regulatory sequences (promoter and terminator). In order to do this, the nucleotide sequence of the gene must be determined and the region coding for the protein must be exactly known.
Unlike yeast, many bacteria can utilize GTG in addition to ATG as a start codon for protein synthesis. Thus, although the gene would be coupled to yeast regulatory sequences, the authentic protein would not be produced in these cases. When the N-terminal amino acid sequence of the protein is not known, as is the case with the .alpha.-ALDC enzymes, the correct N-terminus cannot always be identified from the nucleotide sequence. This is even more difficult if both GTG and ATG can represent a start codon. If DNA sequences of the gene are available from other closely related organisms, homology of the sequences facilitates the localization of the coding region. In any case, the only way to show that a piece of DNA is functional in yeast, is to demonstrate corresponding enzyme activity in yeast cells transformed with this DNA molecule.
When a protein coding region is correctly coupled to yeast regulatory sequences expression of the foreign gene in yeast is sometimes obtained and the corresponding protein is made in some form. However, there is no way to predict the exact production level of the protein. This depends, for instance, on the promoter used, the exact expression construction made, and the copy number of the gene in the transformant. It also depends on the precise nature of each protein. Consequently, some heterologous proteins cannot be produced in active form in yeast (Hollenberg, 1987), some are unstable due to cytoplasmic proteolysis (Stepien et al., 1983; Urdea et al., 1983) and some are produced at a very much lower level than expected (Mellor et al., 1983). Even a low level of expression can be harmful or even toxic to the cell and production of a foreign protein can lead to a very much reduced growth rate of the yeast (Kingsman et al., 1985). When genes involved in the house keeping activities of the cell, like the .alpha.-ald gene involved in the leucine-valine pathway, are expressed, it is impossible to predict the changes caused in the yeast metabolism. E.g. the formation of fusel oils, the main flavour compounds of beer, is linked to the amino acid metabolism of brewer's yeast cells.
Alteration of the amounts of different higher alcohols (fusel oils) has serious effects on the taste of beer. As important, since the enzyme .alpha.-ALDC effectively removes .alpha.-acetolactate from the cellular pool, is that overproduction of the enzyme would make the yeast auxotrophic, in that it would require the addition of isoleucine and valine to the wort. This would naturally disturb the growth of the yeast and thus alter the flavour and alcohol content of the beer. This type of yeast would therefore be useless for production of beer.
In brewing, the character and quality of the final product, beer, is directly dependent on the growth and metabolism of the yeast strain used in the process. Slight changes can dramatically affect the amount of numerous flavour compounds as well as alcohol produced by the brewer's yeast or cause technical problems in the process. The expression of a foreign gene has to be fine-tuned so that the brewing properties of the strain are not altered.
Since the brewer's yeast is a food microbe, it is important that the yeast does not contain any heterologous DNA other than the coding region for the .alpha.-ald gene and that the rearrangements caused to the yeast genome are minimal. One way to achieve this is to integrate the gene to the yeast chromosome. The place of integration has to be considered since integration to some chromosomal loci might cause reduced growth rate or instability of the integrated DNA (Penttila et al., 1987). Stability of the foreign DNA in the strain is important especially in brewing where the yeast is normally recycled in several successive fermentations and where no additional selection pressure can be applied.
The function of the gene, its stability and the effects of the enzyme produced have to be shown in a brewer's yeast strain used normally for production of beer and in conditions (pilot scale) which correspond closely to the actual full scale process. Only these results permit the demonstration that the brewer's yeast producing .alpha.-ALDC can be used for beer production.